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PROSTATE Publication-derived

Castration-Resistant Prostate Cancer Organoids

Full SOP with Academic or a plan

Source Gao et al., 2014 · Memorial Sloan Kettering Cancer Center · 10.1016/j.cell.2014.08.016

Authors: Dong Gao, Ian Vela, Andrea Sboner, Phillip J. Iaquinta, Wouter R. Karthaus, Anuradha Gopalan, Catherine Dowling, Jackline N. Wanjala, Eva A. Undvall, Vivek K. Arora, John Wongvipat, Myriam Kossai, Sinan Ramazanoglu, Luendreo P. Barboza, Wei Di, Zhen Cao, Qi Fan Zhang, Inna Sirota, Leili Ran, Theresa Y. MacDonald, Himisha Beltran, Juan-Miguel Mosquera, Karim A. Touijer, Peter T. Scardino, Vincent P. Laudone, Kristen R. Curtis, Dana E. Rathkopf, Michael J. Morris, Daniel C. Danila, Susan F. Slovin, Stephen B. Solomon, James A. Eastham, Ping Chi, Brett Carver, Mark A. Rubin, Howard I. Scher, Hans Clevers, Charles L. Sawyers, Yu Chen Duration: 56 days 10 phases Cell source: Patient-Derived Prostate Cancer (Advanced/Castration-Resistant)

Abstract

This protocol describes the establishment and culture of patient-derived prostate cancer organoid lines from metastatic biopsies and circulating tumor cells. Seven organoid lines were successfully derived and extensively characterized for genomic alterations, gene expression, and drug sensitivity, representing diverse prostate cancer subtypes including AR-dependent adenocarcinoma, AR-negative adenocarcinoma, neuroendocrine carcinoma, and squamous differentiation.

Cell source
Patient-Derived Prostate Cancer (Advanced/Castration-Resistant)
Application
Disease modeling, Drug screening, Patient-derived tumor models

Protocol overview

37 steps across 10 phases

Phase 1 · Tissue Acquisition and Processing Day 0
  1. a Tissue Biopsy Collection
  2. b Mince Tissue Sample
  3. c Collagenase Digestion
  4. d Cell Washing
Phase 2 · CTC Isolation (when applicable) Day 0
  1. a CD45 Depletion
  2. b Ficoll-Paque Separation
  3. c CTC Washing
Phase 3 · Organoid Seeding and Culture Initiation Day 0-1
  1. a Matrigel Seeding
  2. b Culture in Prostate Organoid Medium
  3. c Monitor Organoid Formation
Phase 4 · Organoid Passaging and Propagation Day 7 onwards (weekly passage)
  1. a Select for Continuous Propagation
  2. b Dissociate Organoids for Passaging
  3. c Passage Organoids
Phase 5 · Genomic Characterization – Sample Preparation Upon establishment of stable lines
  1. a DNA Extraction
  2. b RNA Extraction
Phase 6 · Whole-Exome Sequencing and Analysis Days 1-14 (sequencing and data processing)
  1. a Library Preparation
  2. b Paired-End Sequencing
  3. c Sequence Alignment
  4. d Quality Score Recalibration and Indel Realignment
  5. e Variant Calling
  6. f Somatic Mutation Filtering
Phase 7 · RNA-Sequencing and Expression Analysis Days 1-14 (sequencing and data processing)
  1. a RNA Library Preparation and Sequencing
  2. b Read Alignment and Quantification
  3. c Clustering and Heatmap Analysis
Phase 8 · Array CGH and Copy Number Analysis Days 1-7 (sample processing and analysis)
  1. a Array CGH Preparation
  2. b Segmentation and Copy Number Calling
Phase 9 · In Vitro Drug Sensitivity Testing Days 1-4 (per assay)
  1. a Cell Plating for Viability Assay
  2. b Drug Treatment
  3. c Viability Measurement
  4. d IC50 Determination
Phase 10 · In Vivo Xenograft Studies Days 0-56+ (tumor establishment and treatment)
  1. a Organoid Cell Preparation
  2. b Matrigel Mixing
  3. c Xenograft Implantation
  4. d Tumor Growth Monitoring
  5. e Drug Treatment Initiation
  6. f Tumor Response Assessment
  7. g Tissue Harvest and Histology

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Attribution

This SOP was written by Organthis based on the published method in Gao et al., 2014. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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